Oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum

The oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum was studied under laboratory conditions. After screening, A. brasilense strain SR80 was chosen for further investigation. The strain was capable of degrading 56.5% of crude oil (added in a concentration of 1%) over 14 days in a medium containing malate as an additional source of carbon and energy. Studies of associative properties showed that the strain had positive chemotaxis to wheat root exudates, colonized wheat roots, and produced indolyl-3-acetic acid. The synthesis of indolyl-3-acetic acid was not inhibited by oil. Under hydroponic conditions, crude oil stimulated growth of A. brasilense SR80, which promoted development of the wheat root system in the presence of oil and enhanced the level of oil degradation by the plant-microbial association.     

解淀粉芽胞杆菌HZ-12中ysnE参与吲哚-3-乙酸合成的代谢途径研究

【目的】吲哚-3-乙酸是调控植物生长发育和生理活动的重要激素,吲哚-3-乙酸N-乙酰转移酶YsnE在吲哚-3-乙酸合成中发挥重要作用,本研究拟解析解淀粉芽胞杆菌中YsnE参与吲哚-3-乙酸合成的代谢途径。【方法】通过基因ysnE缺失和强化表达,分析ysnE对吲哚-3-乙酸合成影响,结合吲哚-3-乙酸合成中间物(吲哚丙酮酸、吲哚乙酰胺、色胺和吲哚乙腈)添加和体外酶转化实验,解析ysnE参与吲哚-3-乙酸合成的代谢途径。【结果】明确了YsnE在解淀粉芽胞杆菌HZ-12吲哚-3-乙酸合成中发挥重要作用。发现ysnE缺失菌株中的吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈利用显著降低,揭示了YsnE主要发挥吲哚丙酮酸脱羧酶YclB和吲哚乙酰胺水解酶/腈水解酶/腈水合酶YhcX的功能,并通过参与吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径来影响吲哚-3-乙酸合成。【结论】初步揭示了YsnE通过影响吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径参与吲哚-3-乙酸合成的代谢机理,为吲哚-3-乙酸合成途径解析和代谢工程育种构建吲哚-3-乙酸高产菌株奠定了基础。     

Molecular cloning and sequence analysis of an Azospirilium brasilense indole-3-pyruvate decarboxylase gene

Molecular Genetics and Genomics - Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid...     

Biosynthesis of indole-3-acetic acid in Azospirillum brasilense. Insights from quantum chemistry.

Quantum chemical methods AM1 and PM3 and chromatographic methods were used to qualitatively characterize pathways of bacterial production of indole-3-acetic acid (IAA). The standard free energy changes (delta G(o)'sum) for the synthesis of tryptophan (Trp) from chorismic acid via anthranilic acid and indole were calculated, as were those for several possible pathways for the synthesis of IAA from Trp, namely via indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), and indole-3-acetonitrile (IAN). The delta G(o)'sum for Trp synthesis from chorismic acid was -402 (-434) kJ.mol-1 (values in parentheses were calculated by PM3). The delta G(o)'sum for IAA synthesis from Trp were -565 (-548) kJ.mol-1 for the IAN pathway, -481 (-506) kJ.mol-1 for the IAM pathway, and -289 (-306) kJ.mol-1 for the IPyA pathway. By HPLC analysis, the possibility was assessed that indole, anthranilic acid, and Trp might be utilized as precursors for IAA synthesis by Azospirillum brasilense strain Sp 245. The results indicate that there is a high motive force for Trp synthesis from chorismic acid and for IAA synthesis from Trp, and make it unlikely that anthranilic acid and indole act as the precursors to IAA in a Trp-independent pathway.     

Indole-3-acetic acid catabolism in Zea mays seedlings. Metabolic conversion of oxindole-3-acetic acid to 7-hydroxy-2-oxindole-3-acetic acid 7'-O-beta-D-glucopyranoside

A new metabolite of the plant growth substance indole-3-acetic acid has been extracted from Zea mays seedlings and characterized as the 7'-O-beta-D-glucopyranoside of 7-hydroxy-2-oxindole-3-acetic acid. This compound was the major product formed from [5-3H] 2-oxindole-3-acetic acid, incubated with intact plants or root and coleoptile sections. Identification was by gas chromatography-mass spectrometry of the trimethylsilyl derivative and by analysis of the hydrolysis products. A synthesis is reported for 7-hydroxy-2-oxindole-3-acetic acid. These results and prior work demonstrate the following catabolic route for indole-3-acetic acid in Zea: indole-3-acetic acid----2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid glucoside.     

Key physiological properties contributing to rhizosphere adaptation and plant growth promotion abilities of Azospirillum brasilense

Azospirillum brasilense is a plant growth promoting rhizobacterium (PGPR) that is being increasingly used in agriculture in a commercial scale. Recent research has elucidated key properties of A.?brasilense that contribute to its ability to adapt to the rhizosphere habitat and to promote plant growth. They include synthesis of the auxin indole-3-acetic acid, nitric oxide, carotenoids, and a range of cell surface components as well as the ability to undergo phenotypic variation. Storage and utilization of polybetahydroxyalkanoate polymers are important for the shelf life of the bacteria in production of inoculants, products containing bacterial cells in a suitable carrier for agricultural use. Azospirillum brasilense is able to fix nitrogen, but despite some controversy, as judging from most systems evaluated so far, contribution of fixed nitrogen by this bacterium does not seem to play a major role in plant growth promotion. In this review, we focus on recent advances in the understanding of physiological properties of A.?brasilense that are important for rhizosphere performance and successful interactions with plant roots.     

Characterization of phenylpyruvate decarboxylase, involved in auxin production of Azospirillum brasilense

Azospirillum brasilense belongs to the plant growth-promoting rhizobacteria with direct growth promotion through the production of the phytohormone indole-3-acetic acid (IAA). A key gene in the production of IAA, annotated as indole-3-pyruvate decarboxylase (ipdC), has been isolated from A. brasilense, and its regulation was reported previously (A. Vande Broek, P. Gysegom, O. Ona, N. Hendrickx, E. Prinsen, J. Van Impe, and J. Vanderleyden, Mol. Plant-Microbe Interact. 18:311-323, 2005). An ipdC-knockout mutant was found to produce only 10% (wt/vol) of the wild-type IAA production level. In this study, the encoded enzyme is characterized via a biochemical and phylogenetic analysis. Therefore, the recombinant enzyme was expressed and purified via heterologous overexpression in Escherichia coli and subsequent affinity chromatography. The molecular mass of the holoenzyme was determined by size-exclusion chromatography, suggesting a tetrameric structure, which is typical for 2-keto acid decarboxylases. The enzyme shows the highest kcat value for phenylpyruvate. Comparing values for the specificity constant kcat/Km, indole-3-pyruvate is converted 10-fold less efficiently, while no activity could be detected with benzoylformate. The enzyme shows pronounced substrate activation with indole-3-pyruvate and some other aromatic substrates, while for phenylpyruvate it appears to obey classical Michaelis-Menten kinetics. Based on these data, we propose a reclassification of the ipdC gene product of A. brasilense as a phenylpyruvate decarboxylase (EC 4.1.1.43).     

Wheat inoculation with Azospirillum brasilense Sp6 and some mutants altered in nitrogen fixation and indole-3-acetic acid production

Abstract Inoculation of wheat seedlings with Azospirillum brasilense Sp6 produced an increase in the number and length of the lateral roots as a plant response. Inoculation with a Nif⁻ mutant, A. brasilense SpF103, which is producer of indole-3-acetic acid (IAA), yielded a very similar plant response. However, inoculation with a Nif⁻ mutant, A. brasilense SpF57, which is a low producer of IAA, did not elitic any response from the plant. The data suggest that the root system response of wheat seedlings to bacterial inoculation is due mainly to production of auxin-type substances by the microorganism.     

Tryptophol Formation by Zygosaccharomyces priorianus

Zygosaccharomyces priorianus converted L-tryptophan to tryptophol and to small quantities of indole-3-acetic acid. Neither tryptophol nor indole-3-acetic acid was metabolized when added separately to growing cultures. The possible intermediacy of indole-3-pyruvic acid, indole-3-acetaldehyde, and tryptamine in the degradation of L-tryptophan was tested by feeding these compounds to Z. priorianus and Saccharomyces cerevisiae. Indole-3-pyruvic acid and indole-3-acetaldehyde were converted to tryptophol and indole-3-acetic acid, with the latter accumulating only in small amounts. Tryptamine was converted to its N-acetyl derivative by these organisms. A qualitative study was made on the metabolism of L-phenylalanine, L-tyrosine, and L-5-hydroxytryptophan by these organisms. Like L-tryptophan, these amino acids were metabolized to their respective alcohol and acid derivatives. Of a large number of organisms tested, the yeasts possessed the highest capacity for degrading L-tryptophan to tryptophol.     

Physiological evidence for differently regulated tryptophan-dependent pathways for indole-3-acetic acid synthesis in Azospirillum brasilense

Disruption of ipdC, a gene involved in indole-3-acetic acid (IAA) production by the indole pyruvate pathway in Azospirillum brasilense Sp7, resulted in a mutant strain that was not impaired in IAA production with lactate or pyruvate as the carbon source. A tryptophan auxotroph that is unable to convert indole to tryptophan produced IAA if tryptophan was present but did not synthesise IAA from indole. Similar results were obtained for a mutant strain with additional mutations in the genes ipdC and trpD. This suggests the existence of an alternative Trp-dependent route for IAA synthesis. On gluconate as a carbon source, IAA production by the ipdC mutant was inhibited, suggesting that the alternative route is regulated by catabolite repression. Using permeabilised cells we observed the enzymatic conversion of tryptamine and indole-3-acetonitrile to IAA, both in the wild-type and in the ipdC mutant. IAA production from tryptamine was strongly decreased when gluconate was the carbon source.     

Biosynthesis of auxin by the gram-positive phytopathogen Rhodococcus fascians is controlled by compounds specific to infected plant tissues

The role and metabolism of indole-3-acetic acid in gram-negative bacteria is well documented, but little is known about indole-3-acetic acid biosynthesis and regulation in gram-positive bacteria. The phytopathogen Rhodococcus fascians, a gram-positive organism, incites diverse developmental alterations, such as leafy galls, on a wide range of plants. Phenotypic analysis of a leafy gall suggests that auxin may play an important role in the development of the symptoms. We show here for the first time that R. fascians produces and secretes the auxin indole-3-acetic acid. Interestingly, whereas noninfected-tobacco extracts have no effect, indole-3-acetic acid synthesis is highly induced in the presence of infected-tobacco extracts when tryptophan is not limiting. Indole-3-acetic acid production by a plasmid-free strain shows that the biosynthetic genes are located on the bacterial chromosome, although plasmid-encoded genes contribute to the kinetics and regulation of indole-3-acetic acid biosynthesis. The indole-3-acetic acid intermediates present in bacterial cells and secreted into the growth media show that the main biosynthetic route used by R. fascians is the indole-3-pyruvic acid pathway with a possible rate-limiting role for indole-3-ethanol. The relationship between indole-3-acetic acid production and the symptoms induced by R. fascians is discussed.     

Comparative in situ analysis of ipdC-gfpmut3 promoter fusions of Azospirillum brasilense strains Sp7 and Sp245

Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial production of indole-3-acetic acid in the rhizosphere. Field inoculation experiments had revealed more consistent plant growth stimulation using A. brasilense strain Sp245 as compared with the strain Sp7. Therefore, the in situ expression of the key gene ipdC (indole-3-pyruvate decarboxylase) was examined in these two strains. Within the ipdC promoter of strain Sp245 a region of 150 bases was identified, which was missing in strain Sp7. Thus, three different translational ipdC promoter fusions with gfpmut3 were constructed on plasmid level: the first contained the part of the Sp245 promoter region homologous to strain Sp7, the second was bearing the complete promoter region of Sp245 including the specific insertion and the third comprised the Sp7 promoter region. By comparing the fluorescence levels of these constructs after growth on mineral medium with and without inducing amino acids, it could be demonstrated that ipdC expression in A. brasilense Sp245 was subject to a stricter control compared with strain Sp7. Microscopic detection of these reporter strains colonizing the rhizoplane documented for the first time an in situ expression of ipdC.     

Aerobic nitric oxide production by Azospirillum brasilense Sp245 and its influence on root architecture in tomato

The major feature of the plant-growth-promoting bacteria Azospirillum brasilense is its ability to modify plant root architecture. In plants, nitric oxide (NO) mediates indole-3-acetic acid (IAA)-signaling pathways leading to both lateral (LR) and adventitious (AR) root formation. Here, we analyzed aerobic NO production by A. brasilense Sp245 wild type (wt) and its mutants Faj009 (IAA-attenuated) and Faj164 (periplasmic nitrate reductase negative), and its correlation with tomato root-growth-promoting effects. The wt and Faj009 strains produced 120 nmol NO per gram of bacteria in aerated nitrate-containing medium. In contrast, Faj164 produced 5.6 nmol NO per gram of bacteria, indicating that aerobic denitrification could be considered an important source of NO. Inoculation of tomato (Solanum lycopersicum Mill.) seedlings with both wt and Faj009 induced LR and AR development. In contrast, Faj164 mutant was not able to promote LR or AR when seedlings grew in nitrate. When NO was removed with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), both LR and AR formation were inhibited, providing evidence that NO mediated Azospirillum-induced root branching. These results show that aerobic NO synthesis in A. brasilense could be achieved by different pathways and give evidence for an NO-dependent promoting activity on tomato root branching regardless of bacterial capacity for IAA synthesis.     

Biosynthesis of Auxin by the Gram-Positive Phytopathogen Rhodococcus fascians Is Controlled by Compounds Specific to Infected Plant Tissues

The role and metabolism of indole-3-acetic acid in gram-negative bacteria is well documented, but little is known about indole-3-acetic acid biosynthesis and regulation in gram-positive bacteria. The phytopathogen Rhodococcus fascians, a gram-positive organism, incites diverse developmental alterations, such as leafy galls, on a wide range of plants. Phenotypic analysis of a leafy gall suggests that auxin may play an important role in the development of the symptoms. We show here for the first time that R. fascians produces and secretes the auxin indole-3-acetic acid. Interestingly, whereas noninfected-tobacco extracts have no effect, indole-3-acetic acid synthesis is highly induced in the presence of infected-tobacco extracts when tryptophan is not limiting. Indole-3-acetic acid production by a plasmid-free strain shows that the biosynthetic genes are located on the bacterial chromosome, although plasmid-encoded genes contribute to the kinetics and regulation of indole-3-acetic acid biosynthesis. The indole-3-acetic acid intermediates present in bacterial cells and secreted into the growth media show that the main biosynthetic route used by R. fascians is the indole-3-pyruvic acid pathway with a possible rate-limiting role for indole-3-ethanol. The relationship between indole-3-acetic acid production and the symptoms induced by R. fascians is discussed.     

Involvement of naphthalene dioxygenase in indole-3-acetic acid biosynthesis by Pseudomonas putida

Two variants of plant growth-promoting strain Pseudomonas putida BS1380 harboring the naphthalene degradative plasmid pBS2 and the recombinant plasmid pNAU64 that contains the genes encoding for naphthalene dioxygenase were constructed by conjugation. The ability of this strain to produce phytohormone indole-3-acetic acid from different carbon sources was studied. Indole-3-acetic acid synthesis by these transconjugants was 15-30 times as much in contrast to a wild-type strain with glucose as the sole carbon source. No difference was observed in other carbon or nitrogen sources. It is suggested that naphthalene dioxygenase is involved in the conversion of indole-3-pyruvic acid to indole-3-acetic acid.     

Role of Auxin in Induction of Polarity in Fucus vesiculosus Zygotes

We studied the effects of auxin (indole-3-acetic acid) on formation of the primary polarity axis in zygotes of the brown algae Fucus vesiculosusL. Within the first 2.5 h after fertilization, the zygotes release this phytohormone in the ambient medium. The treatment of developing zygotes with the inhibitor of indole-3-acetic acid transport from the cell 2,3,5-triiodobenzoic acid at 5 mg/l arrests the auxin secretion and leads to its accumulation in the cells. This causes a significant delay in zygote polarization. The treatment of zygotes with the exogenous indole-3-acetic acid at 1 mg/l stimulates cell polarization and formation of a rhizoid protuberance. When auxin was added to the medium with triiodobenzoic acid, the inhibitory effect of the latter was eliminated. It has been proposed that the content of indole-3-acetic acid in the ambient medium is a key factor in the induction of polarity of the F. vesiculosus zygotes.     

Abscission: movement and conjugation of auxin

A 1-hour application of indole-3-acetic acid to bean (Phaseolus vulgaris L. cv. Red Kidney) explants inhibited abscission for an 8-hour aging period. Use of indole-3-acetic acid-¹⁴C showed that the applied indole-3-acetic acid was conjugated within explant tissue and that this conjugation mechanism accounts for loss of effectiveness of indole-3-acetic acid in inhibiting abscission after 8 hours. Reapplication of indole-3-acetic acid to an explant at a later time, before the induced aging requirement was completed reinhibited abscission. 2,4-Dichlorophenoxyacetic acid, which is not destroyed or conjugated by this system, did not lose its ability to inhibit abscission. It was concluded that indole-3-acetic acid destruction is one of the processes involved in the aging stage of abscission in explants.